Field of Endeavor
The present invention relates to chemical amplification and more particularly to for fluid partitioning between polymeric sheets for purposes of chemical amplification, such as Polymerase Chain Reaction (PCR)-based DNA (or RNA) detection, or for other chemical processing/separations.
State of Technology
Since the development by Kary Mullis of the PCR technique for amplification of DNA strands in 1983 for which he received the Nobel Prize for Chemistry in 1993, the chemistry and equipment for PCR have advanced dramatically. Amplification of DNA can be achieved starting from a single strand. In 1987, a joint venture between Perkin-Elmer and Cetus introduced Taq polymerase, a DNA polymerase that is stable through many rounds of thermal cycling. Starting around 1996, with the advent of fluorescent probes, real-time PCR methods have been employed where the amplification of DNA can be monitored as it occurs with optical detection techniques.
PCR is commonly carried out in a reaction volume of 10-200 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. Using this typical approach, PCR consists of a series of 20-40 repeated temperature cycles. Efficiencies have been gained by using arrays of reaction sites, for example arrays of 96 wells on a plate, and techniques for faster heating and cooling of the samples. Although initial efforts at DNA detection required hours to days, these fluorophore-based techniques using arrays of relatively small reaction sites have reduced the time-to-detection to less than one-hour.
More recently, additional time-to-detection advances have been achieved for detection of DNA, which have been demonstrated to be particularly useful for very dilute DNA solutions. When the initial DNA sample is very dilute, partitioning the sample into many separate aliquots can effectively increase the DNA to volume ratio for the fluid partitions that contain DNA, can decrease the number of thermal cycles necessary to reach the minimum fluorescent probe detection limit, and thereby decrease the detection time. Several methods have been employed for this fluid partitioning, including micro-well plates, and arrays of reaction sites and flow channels formed into various substrates. Most of these fluid partitioning approaches have reaction volumes on the order of a micro-liter (μl) and were discrete plates or other batch reaction array approaches. However, some effort has been made to reduce the volume of reaction sites toward the nano-liter range using arrays of very small capillaries, reaction sites on chips, or other droplet techniques.
The most promising recent advance, which not only significantly reduces the sample volume, but can also be implemented as a continuous process providing absolute quantification, is the suspension of micro-liter (i.e., 10−6 liter) to pico-liter (i.e., 10−12 liter) sized droplets in an immiscible carrier fluid. This approach may also be extended to include emulsions or gels. Lawrence Livermore National Laboratory, Quantalife™, and RainDance Technologies™ have developed approaches that involve very small droplets in an immiscible fluid. Quantalife™ has demonstrated the ability to generate mono-disperse nanoliter (i.e. 10−9 liter) sized droplets, perform the thermo-cycling in a conventional 96 well cycler with 20,000 droplet per well, and then use a flow system for detection of individual droplets in series at a rate of 32 wells/hr or 640,000 droplets/hr.
The present invention provides the same advantages as the approach of suspending droplets in an immiscible fluid, including (1) absolute quantification, (2) very small sample size resulting in reduced detection times, and (3) potential for continuous operation for increased throughput, but also includes the additional advantages of (1) providing a robust flexible framework for the partitioned samples that allow more directing handling and facilitates processing in automated equipment, (2) eliminating the need for an immiscible fluid thereby decreasing the time for heating and cooling and the resultant reduction in time to detection, and (3) allowing simultaneous detection of many samples in a row or array, again reducing detection time.
In addition to being applicable for DNA amplification and detection process using thermal cyclers and optical detection, the invention described herein is applicable to isothermal amplification processes and other DNA detection methods.
U.S. Pat. No. 7,041,481 issued May 9, 2006 to Brian L. Anderson, Billy W. Colston, Jr., and Chris Elkin and U.S. Pat. No. RE 41,780 for Chemical amplification based on fluid partitioning contains the state of technology information reproduced below. U.S. Pat. No. 7,041,481 and U.S. Pat. No. RE 41,780 are incorporated herein by this reference for all purposes.
The polymerase chain reaction (PCR), is a cyclic process whereby a large quantity of identical DNA strands can be produced from one original template. The procedure was developed in 1985 by Kary Mullis, who was awarded the 1993 Nobel Prize in chemistry for his work. In PCR, DNA is immersed in a solution containing the enzyme DNA polymerase, unattached nucleotide bases, and primers, which are short sequences of nucleotides designed to bind with an end of the desired DNA segment. Two primers are used in the process: one primer binds at one end of the desired segment on one of the two paired DNA strands, and the other primer binds at the opposite end on the other strand. The solution is heated to break the bonds between the strands of the DNA, then when the solution cools, the primers bind to the separated strands, and DNA polymerase quickly builds a new strand by joining the free nucleotide bases to the primers in the 5′-3′ direction. When this process is repeated, a strand that was formed with one primer binds to the other primer, resulting in a new strand that is restricted solely to the desired segment. Thus the region of DNA between the primers is selectively replicated. Further repetitions of the process can produce a geometric increase in the number of copies, (theoretically 2 n if 100% efficient whereby n equals the number of cycles), in effect billions of copies of a small piece of DNA can be replicated in several hours.
A PCR reaction is comprised of (a) a double-stranded DNA molecule, which is the “template” that contains the sequence to be amplified, (b) primer(s), which is a single-stranded DNA molecule that can anneal (bind) to a complimentary DNA sequence in the template DNA; (c) dNTPs, which is a mixture of dATP, dTTP, dGTP, and dCTP which are the nucleotide subunits that will be put together to form new DNA molecules in the PCR amplification procedure; and (d) Taq DNA polymerase, the enzyme which synthesizes the new DNA molecules using dNTPs.